A method for preparing cell smears and its application

Abstract

The present invention discloses a method for preparing a cell smear, which comprises the steps of preparing a cell suspension, fixing cells, staining cells and drying cells, wherein the cell drying is to culture the cell smear at 50°C under aeration conditions for 40-60 minutes. The present invention also discloses the use of the cell smear in the preparation of a drug for preventing and treating a disease. The preparation method of the present invention can maintain or improve the cell recovery rate, retain the complete cell nucleus type and not affect the membrane protein antibody staining, and can also reduce the drying time and speed up the overall experimental progress.

Description

Technical Field

[0001] The invention belongs to the field of biological and medical detection, and specifically relates to a preparation method of a cell smear and an application thereof. Background Art

[0002] Circulating Tumor Cells (CTCs) refer to solid tumor epithelial cells that fall off from the primary tumor and enter the peripheral blood circulation system during tumor growth. CTCs will attach to distant tissues through the blood circulation system to grow and form new tumor tissues - metastatic foci. A large amount of clinical data shows that tumor metastasis is the direct cause of death in cancer patients. Therefore, early detection of CTCs is of great significance for early diagnosis of cancer, postoperative prognosis, and efficacy evaluation. However, the number of CTCs in peripheral blood is very small, accounting for only 1 / 10 of peripheral blood leukocytes. 6 ~1 / 10 7 , and its number is dynamically distributed in the peripheral blood, and there will be a lag phenomenon, so the requirements for detecting CTCs are very high.

[0003] The detection of CTCs in peripheral blood samples usually includes two steps: enrichment and identification. During enrichment, the white blood cell suspension containing CTCs is first separated from the red blood cells by appropriate centrifugation to prepare for the subsequent screening, staining, drying, fixation and identification of CTCs. Among them, the conditions for drying the cell suspension and fixing the cells are usually an oven at 32°C overnight, and the air is not turned on. However, the long drying time greatly affects the duration of the identification operation, and the cell recovery rate is not ideal. "Clinical Study on the Prediction of Microvascular Invasion of Hepatocellular Carcinoma by Circulating Tumor Cell Detection" (Master's Thesis of the Second Military Medical University, author: Wang Yu, tutor: Shi Lehua) discloses a method for preparing a cell smear, in which the cells are first dried and then fixed, and the drying time is short, only 15-30 minutes, but the cell recovery rate is only 52.41%. Therefore, there is currently a lack of a method for preparing a cell smear for CTCs detection that can maintain or improve the cell recovery rate, does not affect the cell nucleus type and the cell morphology after membrane protein antibody staining, and has a short operation time. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a cell smear and its application, in view of the lack of a cell smear method in the prior art that can maintain or improve the cell recovery rate, retain the complete cell nucleus without affecting the membrane protein antibody staining, and can also reduce the drying time and speed up the overall experimental progress.

[0005] To solve the above technical problems, one of the technical solutions of the present invention is to provide a method for preparing a cell smear, which includes the steps of preparing a cell suspension, fixing cells, staining cells and drying cells, wherein the cell drying is to culture the cell smear at 50° C. under aeration conditions for 40-60 minutes. In the art, the above cell drying process is also called "baking".

[0006] In addition to being able to control the culture temperature under different conditions, some constant temperature incubators in the art also have a blast function, so the above-mentioned "blast condition" refers to turning on the blast function of such constant temperature incubator. Theoretically, all constant temperature incubators with a blast function can be used in the present invention. In a preferred specific embodiment of the present invention, an electric constant temperature drying oven (model: 101) of Shanghai Yetuo Instrument Co., Ltd. is used. Under the premise that other parameters in the technical solution of the present invention remain unchanged, an electric constant temperature drying oven with the same or nearly the same blast function (including wind speed) as the electric constant temperature drying oven of the present invention can achieve the effect of the present invention.

[0007] In a preferred embodiment, in the method for preparing the cell smear, the cell fixing step is before the cell drying step. The cell fixing step in the prior art is after the cell drying step, and its cell recovery rate is lower than that of the present invention, which is only 52.41%.

[0008] In a preferred embodiment, in the method for preparing the cell smear, the incubation time under the aeration condition is more than 50 minutes; for example, 50 minutes. Under the same other conditions, a 50-minute baking time can bring the best cell recovery rate (72.99±5.37%).

[0009] In a preferred embodiment, in the preparation method of the cell smear, the fixative used for the cell fixation is a paraformaldehyde fixative. Conventional cell fixatives in the art can theoretically be used in the present invention. Paraformaldehyde is a good fixative for a variety of tissue cells, and is superior to other fixatives for the preservation of lipids and enzymes. In addition, the autofluorescence of paraformaldehyde is very weak and will not affect the fluorescence expression of immunofluorescence antibodies. In a more preferred embodiment of the present invention, a 4% paraformaldehyde solution is used, and the % is a weight volume percentage.

[0010] In a preferred embodiment, in the method for preparing the cell smear, the concentration of the cell suspension is 5×10 4 Pieces / mL.

[0011] The cell suspension can be prepared by various conventional buffers in the art. In a preferred embodiment, in the preparation method of the cell smear, the cell suspension is a PBS cell suspension. Preferably, the volume percentage of the 4% paraformaldehyde fixative and the PBS cell suspension is 1:1.

[0012] In a preferred embodiment, in the method for preparing the cell smear, the cells are mammalian cells, preferably human cells, more preferably human blood cells; most preferably, before the step of preparing the cell suspension, the step of separating the human blood cells from the plasma is also included. Separating the human blood cells from the plasma is beneficial to the staining of the cells and the subsequent observation and detection.

[0013] In a preferred specific embodiment, in the preparation method of the cell smear, the cell staining uses CD45 antibody staining solution and / or DAPI staining solution. The CD45 antibody staining solution is preferably a human CD45 antibody staining solution. CD45 is a common antigen on the surface of leukocytes. Generally, leukocytes have this antigen. The human CD45 antibody staining solution has a specific staining effect on blood cells, especially leukocytes. DAPI staining solution is 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), which is a fluorescein that can strongly bind to DNA and is commonly used for fluorescence microscopy observations. DAPI can penetrate the intact cell membrane and is commonly used for staining living cells and fixed cells.

[0014] In a preferred embodiment, the method for preparing the cell smear further comprises the steps of washing with PBS buffer and treating with anhydrous ethanol before adding the DAPI staining solution. Preferably, after the anhydrous ethanol treatment, the method further comprises the step of drying.

[0015] In order to solve the above technical problems, the second technical solution of the present invention is: to provide an application of a cell smear in the preparation of a drug for preventing and treating a disease. Preferably, the disease is cancer; more preferably, the cancer is one or more of malignant B-cell lymphoma, including acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), liver cancer, adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, non-small cell carcinoma, gallbladder cancer, esophageal cancer, melanoma, pancreatic cancer, urothelial carcinoma, head and neck cancer and prostate cancer.

[0016] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0017] The reagents and raw materials used in the present invention are commercially available.

[0018] The positive and progressive effects of the present invention are:

[0019] While greatly shortening the drying time, it also improves the cell recovery rate. The cells enriched in the sample can be retained on the slide to a greater extent, and the cell nucleus and overall cell morphology are kept intact, and membrane protein antibody staining is not affected, which greatly improves the experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The cell staining pictures under different drying temperatures and drying times;

[0021] Figure 2 The cell staining images are at different drying temperatures and the same drying time.

[0022] Figure 3 The figures are the results of cell smears followed by CD45 antibody cell immunofluorescence staining, wherein A is the result of the present invention with air blowing at 50° C. and drying for 60 minutes; and B is the result of the cell smear prepared by the prior art method. DETAILED DESCRIPTION

[0023] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0024] Miniature desktop vacuum pump: Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0025] High-speed centrifuge: ThermoFisher Scientific

[0026] Electric constant temperature drying oven (model: 101): Shanghai Yetuo Instrument Co., Ltd.

[0027] Vortex mixer: Haimen Kylin-Bell Lab Instruments

[0028] Fluorescence scanning microscope: Carl Zeiss

[0029] Density gradient separation medium: Ficoll-Pague, GE Healthcare

[0030] 4% paraformaldehyde solution: Kingmorn

[0031] CD45 Antibody Staining Solution: Biolegend

[0032] PBS buffer: Corning

[0033] Trypan blue dye: Sigma-Aldrich

[0034] Anhydrous ethanol: Sinopharm Chemical Reagent Co., Ltd.

[0035] DAPI staining solution: Shanghai Yisheng Biotechnology Co., Ltd.

[0036] Preparation Example 1 Preparation of cell smear

[0037] 1. Separation of red blood cells and plasma

[0038] 1) Take 6 mL of peripheral blood collected from healthy subjects, gently invert the blood collection tube to mix, and centrifuge at 200 g for 15 minutes at room temperature;

[0039] 2) After centrifugation, the supernatant (i.e., plasma) was discarded using a vacuum pump, and PBS buffer was added to a volume of 6 mL. The blood collection tube was gently inverted to mix;

[0040] 3) Add 3 mL of density gradient separation solution to the labeled 50 mL centrifuge tube A;

[0041] 4) Then slowly add the blood cell mixture from step 2) above to the top of the separation solution in centrifuge tube A, and centrifuge at 400g for 30 minutes at room temperature;

[0042] 5) After centrifugation, the liquid in centrifuge tube A is divided into four layers, namely, a yellow liquid layer, a white film layer, a transparent or light red liquid layer, and a dark brown-red liquid layer from top to bottom. First aspirate the white film layer and then aspirate all the liquid above the dark brown-red liquid layer, and add the liquids to the 50 mL centrifuge tube B in sequence;

[0043] 6) Add PBS buffer to 45 mL, centrifuge at 500 g for 5 minutes at room temperature after balancing, and discard the supernatant to 500 μL;

[0044] 7) Add PBS buffer to 45 mL, centrifuge at 400 g for 5 minutes at room temperature after balancing, and discard the supernatant to 100 μL.

[0045] 2. Cell Fixation

[0046] 1) Pipette 100 μL of 4% paraformaldehyde solution and add it to 100 μL of cell suspension, and gently shake it with a vortex mixer to fix it for 15 minutes;

[0047] 2) Add PBS buffer to 45 mL, centrifuge at 500 g for 5 minutes at room temperature after balancing, and discard the supernatant to 500 μL;

[0048] 3) Add PBS buffer to 45 mL, centrifuge at 400 g for 5 minutes at room temperature after balancing, and discard the supernatant to 100 μL.

[0049] 3. Cell Staining

[0050] 1) Add 1 μL CD45 antibody staining solution to 100 μL cell suspension, incubate at room temperature in the dark for 20 minutes, and gently mix once every 10 minutes using a vortex mixer;

[0051] 2) Add PBS buffer to 45 mL, centrifuge at 500 g for 5 minutes at room temperature after balancing, and discard the supernatant to 100 μL.

[0052] 4. Cell Counting and Drying

[0053] 10 μL was taken from 100 μL of cell suspension, and diluted 10 times with PBS buffer, and an equal volume of trypan blue staining solution was added for staining, and the cells were counted using a cell counter.

[0054] According to the counting results and the experimental sample volume requirements, an appropriate amount of cell suspension and PBS buffer in tube B were taken to reconstitute a concentration of 5×10 4 / mL of cell suspension.

[0055] 200 μL of cell suspension was added to the sample frame of the slide and placed in an oven under different drying conditions for drying. The different drying conditions included: (1) a forced air drying experimental group of 50°C for 50 min, 55°C for 40 min, and 60°C for 30 min and a control group of 32°C overnight drying; and (2) a forced air drying experimental group of 50°C, 55°C, and 60°C for different drying times.

[0056] 5. Cell Identification

[0057] 1) After drying, remove the slide and gently drip 200 μL PBS buffer along the inner corner of the specimen frame, then immediately discard it by aspiration; repeat twice in total;

[0058] 2) Gently drip 200 μL PBS buffer along the inner corner of the specimen frame, let it stand for 2 minutes, and then discard it by aspiration; repeat twice;

[0059] 3) Gently drip 200 μL of anhydrous ethanol along the inner corner of the specimen frame, then immediately discard it by aspiration; repeat twice in total;

[0060] 4) Place the slide in a staining jar filled with anhydrous ethanol and let it stand for 2 minutes. Then take out the slide and place it upright on filter paper to completely absorb the residual liquid from the slide. Use a micro hair dryer to gently blow dry the slide.

[0061] 5) Take 10 μL of DAPI staining solution and drop it in the center of the specimen frame. After placing the cover slip, fix the cover slip to prevent it from sliding. Use a vacuum pump tip or filter paper to squeeze and absorb the excess liquid, and seal the specimen.

[0062] 6) Observe immediately under a fluorescence scanning microscope or store at 4°C away from light.

[0063] Effect Example 1 Cell recovery under the fastest drying conditions

[0064] In order to speed up the drying process as much as possible and find the fastest drying time for the cell suspension, the inventors set up 50min (50℃), 40min (55℃) and 30min (60℃) wind-blown experimental groups (i.e., groups 2-1, 3-1, and 4-1 in Table 1 below), and used 32℃ overnight drying as the control group (i.e., group 1-1). The experiment was repeated three times, with three groups in parallel each time. By counting and calculating the cells treated in the early stage, a drop of about 1×10 4 The cell suspension of 10 cells was then placed in an oven at 50°C, 55°C and 60°C under blast conditions for drying. After the time was up, it was taken out and counted under a microscope after post-processing. The statistical results of cell recovery are shown in Table 1. The nuclear type and membrane protein antibody staining of some cells are shown in Table 1. Figure 1 shown.

[0065] Table 1 Comparison of cell recovery rates under different drying conditions and existing technologies

[0066]

[0067] Note: The letters a and b in the cell recovery rate column indicate whether there are significant differences between groups. There are no significant differences between groups with the same letters, and there are significant differences between groups with different letters.

[0068] As can be seen from Table 1, under the conditions of drying temperature of 50°C, drying time of 50 min, and air blowing on, the cell recovery rate of the experimental group 2-1 was significantly different from that of the control group 1-1, while the cell recovery rates under other drying conditions were not significantly different from those of the control group.

[0069] from Figure 1 It can be seen that the cell nucleus morphology of each experimental group is good without obvious deformation, but the outer red circle of the cell membrane (CD45 antibody expression is positive) of group 3-1 at 55℃, 40min, air blowing on and group 4-1 at 60℃, 30min, air blowing on is obviously missing and deformed, while the outer red circle of the cell membrane of group 2-1 at 50℃, 50min, air blowing on is intact.

[0070] The above results show that drying for 50 minutes at a drying temperature of 50°C with the air blower on can achieve a higher cell recovery rate than drying overnight at 32°C with the air blower off in the prior art, and the cell nucleus type and membrane protein antibody staining effects are good.

[0071] Effect Example 2 Cell recovery after drying for 1 hour

[0072] Under the premise that the drying time was controlled within 1 hour, in order to understand the relationship between drying temperature and cell recovery rate, the inventors compared the cell recovery rate under the shortest drying time and drying for 1 hour at different drying temperatures, and used 32°C overnight drying as the control group. The experiment was repeated three times, with three groups in parallel each time. Similarly, by counting and calculating the cells treated in the early stage, a drop of about 1×10 4 The cell suspension of 10 cells was then placed in an oven at a specific temperature and blast conditions as shown in Table 2 for drying. After 1 hour, it was taken out and counted under a microscope after post-processing. The statistical results of cell recovery are shown in Table 2. The nuclear type and membrane protein antibody staining of some cells are shown in Table 2. Figure 2 shown.

[0073] Table 2 Cell recovery under different temperature and time conditions

[0074]

[0075] Note: The letters a, b, and c in the cell recovery rate column indicate whether there are significant differences between the combinations of drying temperature and drying time. The same letters indicate no significant differences between the combinations.

[0076] It can be seen from Table 2 that under the conditions of the shortest drying time and drying for 1 hour, there was no significant difference in cell recovery rate between the experimental groups: 2-1 and 2-2 (50 min vs. 60 min), 3-1 and 3-2 (40 min vs. 60 min), and 4-1 and 4-2 (30 min vs. 60 min).

[0077] The control group was then dried overnight at 32°C without wind, and the experimental group was dried at various temperatures for 1 hour. The statistical data are shown in Table 3.

[0078] Table 3 Cell recovery rate under different temperature and aeration conditions

[0079]

[0080] Note: The letters a, b, and c in the cell recovery rate column indicate whether there are significant differences between groups. There are no significant differences between groups with the same letters, and there are significant differences between groups with different letters.

[0081] Table 3 shows that there are significant differences in the cell recovery rates of experimental group 2-2 at 50°C with air blast on and experimental group 3-2 at 55°C with air blast on compared with control group 1-1, and the cell recovery rate of experimental group 2-2 at 50°C with air blast on is the highest.

[0082] Figure 2 The results showed that the cell nuclei of each experimental group had good morphology without obvious deformation. The outer red circle of the cell membrane (positive expression of CD45 antibody) of experimental group 3-2 at 55℃, 60min, air blowing on and experimental group 4-2 at 60℃, 60min, air blowing on was severely deformed, even radially. In contrast, the outer cell membrane morphology of experimental group 2-2 at 50℃, 50min, air blowing on was compact and complete.

[0083] The above results show that drying for 50-60 minutes at a drying temperature of 50°C with the air blower on can achieve a higher cell recovery rate than drying overnight at 32°C with the air blower off, and the cell nucleus and membrane protein antibody staining effects are good.

[0084] Comparative Example 1

[0085] According to the conditions in Effect Example 2, 50° C. was taken as the drying temperature, and the drying time was extended to 70 minutes, 80 minutes and 90 minutes under the blast condition. The results are shown in Table 4 below:

[0086] Table 4 Cell recovery rate at different drying times

[0087]

[0088] According to the results shown in Table 4, when the drying time is extended to more than 60 minutes, the cell recovery rate decreases significantly. That is, under the premise of other parameters being fixed, extending the drying time does not always increase the cell recovery rate, but has a certain temperature limit.

[0089] Comparative Example 2

[0090] "Clinical Study on Detection of Circulating Tumor Cells to Predict Microvascular Invasion of Hepatocellular Carcinoma" (Master's Degree Thesis of the Second Military Medical University, author: Wang Yu, tutor: Shi Lehua) page 19 records a method for making a cell smear, wherein the slide is first baked and then fixed with paraformaldehyde, the volume of the cell suspension added to the slide is 50 μL, the baking temperature is 52°C, and the baking time is 15-30 minutes. The cell recovery rate of the cell picture made according to the method in the article is 52.41%, which is lower than the cell recovery rate of each condition in Example 1 and 2.

[0091] The results of the cell images prepared under the conditions of 50°C forced air drying for 60 minutes in Example 2 of the present invention and the results of the cell immunofluorescence staining with CD45 antibody obtained by the methods of "6. Cell image preparation process" and "7. Immunofluorescence staining preparation process" on page 19 of the article are as follows: Figure 3 As shown in A-3B.

[0092] Figure 3 A-3B shows that the results of cell immunofluorescence staining of CD45 antibody prepared according to the method of the present invention are clearer, such as Figure 3 As shown in A. The same cell immunofluorescence staining results produced according to the method disclosed in the above paper have more serious background staining, such as Figure 3 As shown in B, there is a serious interference in the determination of CD45 positive cells, which is not conducive to obtaining reliable conclusions.

Claims

1. A method for preparing a cell smear, comprising the steps of preparing a cell suspension, fixing cells, staining cells and drying cells, characterized in that: The cell fixing step is before the cell drying step; the cell drying step is to culture the cell smear at 50° C. under aeration conditions for 50-60 minutes; The cells are white blood cells containing CTCs separated from human blood cells after centrifugation; the cells are stained using CD45 antibody staining solution and DAPI staining solution.

2. The method for preparing a cell smear according to claim 1, characterized in that: The culture time under the aeration condition is 50 minutes.

3. The method for preparing a cell smear according to claim 1, characterized in that: The fixative used for cell fixation is paraformaldehyde fixative.

4. The method for preparing a cell smear according to claim 3, characterized in that: The fixative used for cell fixation is 4% paraformaldehyde fixative, where % refers to weight volume percentage.

5. The method for preparing a cell smear according to claim 1, characterized in that: The concentration of the cell suspension was 5×10 4 Pieces / mL.

6. The method for preparing a cell smear according to claim 1, characterized in that: The cell suspension is PBS cell suspension.

7. The method for preparing a cell smear according to any one of claims 1 to 6, characterized in that: The fixative used for cell fixation is 4% paraformaldehyde fixative; the cell suspension is PBS cell suspension; the volume percentage of the 4% paraformaldehyde fixative and the PBS cell suspension is 1:

1.

8. The method for preparing a cell smear according to claim 1, characterized in that: Before the step of preparing the cell suspension, the method further includes the step of separating human blood cells from plasma.

9. The method for preparing a cell smear according to claim 1, characterized in that: The CD45 antibody staining solution is a human CD45 antibody staining solution.

10. The method for preparing a cell smear according to claim 9, characterized in that: Before adding the DAPI staining solution, the steps of washing with PBS buffer and treating with anhydrous ethanol are also included.

11. The method for preparing a cell smear according to claim 10, characterized in that: After the anhydrous ethanol treatment, the method further comprises a drying step.

12. Use of the method for preparing a cell smear as claimed in claim 1 in preparing medicines for preventing and treating diseases.

13. The use according to claim 12, characterized in that The disease is cancer.

14. The use according to claim 13, characterized in that The cancer is one or more of acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma, liver cancer, lung cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, melanoma, pancreatic cancer, urothelial carcinoma, head and neck cancer and prostate cancer.

15. The use according to claim 14, characterized in that The cancer is one of mantle cell lymphoma, colon cancer and non-small cell carcinoma.

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